Zhai et al. Virology Journal (2016) 13:136 DOI 10.1186/s12985-016-0591-6

RESEARCH Open Access Occurrence and sequence analysis of porcine in southern China Shao-Lun Zhai1†, Wen-Kang Wei1†, Xiao-Peng Li1†, Xiao-Hui Wen1, Xia Zhou1, He Zhang1, Dian-Hong Lv1*, Feng Li2,3 and Dan Wang2*

Abstract Background: Following the initial isolation of porcine (PDCoV) from pigs with diarrheal disease in the United States in 2014, the has been detected on swine farms in some provinces of China. To date, little is known about the molecular epidemiology of PDCoV in southern China where major swine production is operated. Results: To investigate the prevalence of PDCoV in this region and compare its activity to other enteric disease of swine caused by porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis (TGEV), and porcine rotavirus group C (Rota C), 390 fecal samples were collected from swine of various ages from 15 swine farms with reported diarrhea. Fecal samples were tested by reverse transcription-PCR (RT-PCR) that targeted PDCoV, PEDV, TGEV, and Rota C, respectively. PDCoV was detected exclusively from nursing piglets with an overall prevalence of approximate 1.28 % (5/390), not in suckling and fattening piglets. Interestingly, all of PDCoV-positive samples were from 2015 rather than 2012–2014. Despite a low detection rate, PDCoV emerged in each province/region of southern China. In addition, compared to TGEV (1.54 %, 5/390) or Rota C (1.28 %, 6/390), there were highly detection rates of PEDV (22.6 %, 88/390) in those samples. Notably, all five PDCoV-positive piglets were co-infected by PEDV. Furthermore, phylogenetic analysis of spike (S) and nucleocapsid (N) gene sequences of PDCoVs revealed that currently circulating PDCoVs in southern China were more closely related to other Chinese strains of PDCoVs than to those reported in United States, South Korea and Thailand. Conclusions: This study demonstrated that PDCoV was present in southern China despite the low prevalence, and supported an evolutionary theory of geographical clustering of PDCoVs. Keywords: Porcine deltacoronavirus, Occurrence, Spike gene, Nucleocapsid gene, Sequence analysis, Southern China

Background (PEDV), transmissible gastroenteritis virus (TGEV), and Before 2012, the subfamily Coronavirinae included three porcine respiratory coronavirus (PRCV) belong to the genus genera (, and Gamma- Alphacoronavirus; meanwhile, porcine hemagglutinating en- coronavirus). However, in 2012, an emerging genus, Delta- cephalomyelitis virus (PHEV) and Porcine deltacoronavirus coronavirus, was found in many animal species including (PDCoV) are assigned to the genus Betacoronavirus and swine from Hong Kong [1]. At present, more than five the genus Deltacoronavirus, respectively [1]. Numerous different have been described in swine pop- studies have shown that more than half of porcine corona- ulations. Among them, porcine epidemic diarrhea virus (including PDCoV) were enteropathogenic and caused acute diarrhea and vomiting in pigs, which resulted * Correspondence: [email protected]; [email protected] – † in huge economic losses for the global swine industry [2 6]. Equal contributors Currently, PDCoV has been reported in Hong Kong, 1Animal Disease Diagnostic Center, Institute of Animal Health, Guangdong Academy of Agricultural Sciences, Guangdong Key Laboratory of Animal North America, Mexico, South Korea, Thailand and some Disease Prevention, Guangdong Open Laboratory of Veterinary Public Health, provinces of China [1, 7–19]. Despite recent progress, Guangzhou 510640, China little is known about the prevalence and epidemiology of 2Department of Biology and Microbiology, South Dakota State University, Brookings, SD 57007, USA PDCoV in southern China (including Guangdong Full list of author information is available at the end of the article

© 2016 The Author(s). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Zhai et al. Virology Journal (2016) 13:136 Page 2 of 11

province, Hainan province, and Guangxi autonomous previously published methods [8]. For amplification of the region), where major swine production is operated. full-length S and N genes, previously reported RT-PCR Therefore, the aim of this study was to investigate the primers (PDCoV-SF2: 5’-AGCGTTGACACCAACCTA prevalence and sequence properties of PDCoV in this TT-3’ and PDCoV-SR2: 5’-TCGTCGACTACCATTCCT region. TAAAC-3’; PDCoV-NF1 : 5’-CCATC GCTCCAAG TC ATTCT-3’ and PDCoV-NR1: 5’-TGGGTGGGTTTAA Methods CAGACATAG-3’) were used. PCR was carried out at Sampling 50 °C 30 min and 95 °C for 5 min, followed by 40 cycles A total of 390 fecal samples (Table 1) were collected of 98 °C for 10 s, 55 °C for 15 s, and 68 °C for 5 and 2 min from 15 commercial swine farms with reported diarrhea for S and N genes, respectively; final extension was per- in southern China. Farms A, D, E, J-O were from formed at 68 °C for 15 min. Positive amplicons were Guangdong province, Farms B, F, G were from Hainan cloned into the pGM-19 T vector (Tiangen Inc. Beijing). province, and farms C, H, I were the Guangxi autono- Furthermore, all positive recombinant plasmids were sub- mous region. Farms A-I derived 30 samples with the mitted to a sequencing company (The Beijing Genomics following arrangement: ten samples from suckling Institute, BGI) and sequenced at least three times. Five S piglets (<3 weeks old), ten samples from nursing piglets gene sequences and five N gene sequences were obtained (between 3 and 9 weeks old), and ten samples from (Additional file 1: Table S1), and have been submitted fattening piglets (>9 weeks old). The samples of farms to GenBank database (accession numbers KU204694- A-I were collected between July and August 2015 and KU204701, KX534090-KX534091). stored at −80 °C until further use. However, the samples of farms J-O were archived samples from 2012 to 2014. Phylogenetic analysis of the S and N genes Prior to viral RNA extraction, fecal samples were diluted Sequence alignment analysis was performed using the one time using Phosphate Buffered Saline (PBS) (pH: Clustal W program implemented in DNAStar software. A 7.4). The supernatants were then collected by centrifuga- was then constructed by the neighbor- tion at 5000 × g for 5 min. 200 μl of clarified superna- joining method using the Molecular Evolutionary Genetics tants was used to extract viral RNA following the Analysis (MEGA) software version 5.1 with 1000 bootstrap manufacturer’s recommendations (Axygen Scientific replications set at 1000. Moreover, the possible recombin- Inc.). RNA samples were stored at −80 °C until further ation event was evaluated in the S and N genes by recom- analysis. bination detection program (RDP) 3.34 software.

Reverse transcription polymerase chain reaction (RT-PCR) Results detection PDCoV detection To detect PDCoV in collected fecal samples, A total of 390 pig fecal samples, collected from 15 swine the previously reported RT-PCR primers (41 F: 5’-TTT farms with reported diarrhea in southern China, were CAGGTGCTCAAAGCTCA-3’ and 735R: 5’-GCGAAA assessed for the presence of PDCoV and other viral en- AGCATTTCCTGAAC-3’) targeting the nucleocapsid teric pathogens (PEDV, TGEV, and Rota C) by RT-PCR. (N) gene with reaction conditions (50 °C for 30 min As summarized in Table 1, the PDCoV genome was de- and 95 °C for 15 min for the reverse transcription reac- tected in specimens from 4 of 15 swine farms. Interest- tion, followed by 40 cycles of PCR amplification at 95 °C ingly, the PDCoV genome was detected only in nursing for 15 s, 55 °C for 45 s, and 72 °C for 1 min, with a final piglets, and was absent in suckling piglets and fattening extension at 72 °C for 7 min) were used [15]. In addition, pigs. Although PDCoV was detected in each province/ molecular detection of the three diarrhea-related enteric region of southern China, its overall prevalence in the viruses (Porcine epidemic diarrhea virus, PEDV; Porcine investigated pigs of various age groups (n =390) was transmissible gastroenteritis virus, TGEV; Porcine rota- relatively low (5/390, 1.28 %). The positive rate could be virus group C, Rota C) was performed in accordance with higher if only nursing piglets were included (5/150, previous methods [20–22] for further evaluation of the 3.33 %). In contrast, the prevalence of PEDV, another por- possible co-infection status with PDCoV in investigated cine coronavirus causing epidemic diarrhea, was relatively pig samples. higher (22.6 %, 88/390). In addition, PEDV was different from PDCoV in that it distributed similarly between nurs- Amplification of the spike (S) and N genes ing (36/150, 24 %) and suckling piglets (47/150, 31.3 %). To perform in-depth sequence comparison and phylogen- Five fattening pigs from farms A, D, F and I were also etic analysis with known reference sequences (Additional tested positive for the PEDV genome. We also examined file 1: Table S1), the complete spike (S) and N genes of whether pigs with diarrhea harbored other enteric viruses PDCoV-positive samples were amplified according to such as TGEV and Rota C. Our results showed that the Zhai et al. Virology Journal (2016) 13:136 Page 3 of 11

Table 1 Sample information and RT-PCR detection results of four diarrhea-associated viruses in pigs of various ages from 15 swine farms in southern China Farm Pigs (Age) Number (n) PDCoV PEDV TGEV Rota C A Suckling piglets (<3 weeks old) 10 0/10 2/10 0/10 0/10 Nursery pigs (>3 weeks old, <9 weeks old) 10 2/10 2/10 0/10 0/10 Fattening pig (>9 weeks old) 10 0/10 1/10 0/10 0/10 B Suckling piglets (<3 weeks old) 10 0/10 3/10 0/10 0/10 Nursery pigs (>3 weeks old, <9 weeks old) 10 1/10 2/10 0/10 0/10 Fattening pig (>9 weeks old) 10 0/10 0/10 0/10 0/10 C Suckling piglets (<3 weeks old) 10 0/10 3/10 0/10 0/10 Nursery pigs (>3 weeks old, <9 weeks old) 10 1/10 3/10 0/10 0/10 Fattening pig (>9 weeks old) 10 0/10 0/10 0/10 0/10 D Suckling piglets (<3 weeks old) 10 0/10 2/10 0/10 0/10 Nursery pigs (>3 weeks old, <9 weeks old) 10 0/10 1/10 0/10 2/10 Fattening pig (>9 weeks old) 10 0/10 1/10 1/10 0/10 E Suckling piglets (<3 weeks old) 10 0/10 1/10 0/10 0/10 Nursery pigs (>3 weeks old, <9 weeks old) 10 1/10 2/10 0/10 0/10 Fattening pig (>9 weeks old) 10 0/10 0/10 0/10 0/10 F Suckling piglets (<3 weeks old) 10 0/10 3/10 0/10 0/10 Nursery pigs (>3 weeks old, <9 weeks old) 10 0/10 4/10 0/10 0/10 Fattening pig (>9 weeks old) 10 0/10 1/10 2/10 0/10 G Suckling piglets (<3 weeks old) 10 0/10 5/10 0/10 0/10 Nursery pigs (>3 weeks old, <9 weeks old) 10 0/10 3/10 0/10 0/10 Fattening pig (>9 weeks old) 10 0/10 0/10 0/10 0/10 H Suckling piglets (<3 weeks old) 10 0/10 7/10 0/10 0/10 Nursery pigs (>3 weeks old, <9 weeks old) 10 0/10 2/10 0/10 0/10 Fattening pig (>9 weeks old) 10 0/10 0/10 0/10 0/10 I Suckling piglets (<3 weeks old) 10 0/10 4/10 0/10 0/10 Nursery pigs (>3 weeks old, <9 weeks old) 10 0/10 2/10 0/10 0/10 Fattening pig (>9 weeks old) 10 0/10 2/10 0/10 0/10 J Suckling piglets (<3 weeks old) 10 0/10 4/10 0/10 0/10 Nursery pigs (>3 weeks old, <9 weeks old) 10 0/10 3/10 0/10 0/10 K Suckling piglets (<3 weeks old) 10 0/10 5/10 0/10 2/10 Nursery pigs (>3 weeks old, <9 weeks old) 10 0/10 5/10 2/10 0/10 L Suckling piglets (<3 weeks old) 10 0/10 2/10 0/10 0/10 Nursery pigs (>3 weeks old, <9 weeks old) 10 0/10 0/10 0/10 0/10 M Suckling piglets (<3 weeks old) 10 0/10 2/10 0/10 0/10 Nursery pigs (>3 weeks old, <9 weeks old) 10 0/10 3/10 0/10 0/10 N Suckling piglets (<3 weeks old) 10 0/10 2/10 0/10 1/10 Nursery pigs (>3 weeks old, <9 weeks old) 10 0/10 1/10 0/10 0/10 O Suckling piglets (<3 weeks old) 10 0/10 2/10 0/10 0/10 Nursery pigs (>3 weeks old, <9 weeks old) 10 0/10 3/10 1/10 0/10 Total 390 5/390 88/390 6/390 5/390 low detection rates (1.54 %, 5/390 for TGEV vs 1.28 %, PDCoV and PEDV was observed (Table 1). All PDCoV 6/390 for Rota C) of the two pathogens were present in positive nursing piglets were also tested positive for PEDV, those pig samples. Intriguingly, co-infection of pigs by thereby indicating a 100 % co-infection rate. Zhai et al. Virology Journal (2016) 13:136 Page 4 of 11

Sequence comparison and phylogenetic analysis of the S possible recombinant events occurring at the N gene of gene of PDCoVs those PDCoV strains. The full-length sequences of S genes in five PDCoV- positive samples from the four different farms were Discussion amplified and designated provisionally CH/GD01/2015, Epidemiology of PDCoVs CH/GD02/2015, CH/GD03/2015, CH/HN01/2015, and PDCoV was first identified by Deltacoronavirus specific- CH/GX01/2015, respectively. Sequencing results showed PCR in rectal swabs of pigs (10.1 %, 17/169) with un- that they were composed of 3480 nucleotides (nt). Com- known healthy status in Hong Kong [1]. Then, PDCoV pared to all published American and individual Asian emerged in United States, China, South Korea and strains (including HKU15-44, CHN-AH-2004, KNU14- Thailand [5, 17, 18]. In most of studies, excluding PEDV, 04, PDCoV/Swine/Thailand/S5015L/2015 and PDCoV/ TGEV and porcine rotavirus, PDCoV, as an important Swine/Thailand/S5011/2015), a 3-nt deletion in the S enteric pathogen, was detected in clinical samples from gene was identified in the five current PDCoV strains, six pigs with diarrhea [9–12, 23]. In addition, it was con- other Chinese viral strains and one Thai viral strain reported firmed experimentally that less than two-week old piglets previously (Additional file 1: Table S1) [5, 17, 18]. Sequence were susceptible to PDCoV, which caused mild to moder- alignment results revealed that CH/GD01/2015, CH/GD02/ ate diarrhea as well as macroscopic and microscopic lesions 2015, CH/GD03/2015, CH/HN01/2015 and CH/GX01/ in small intestines of conventional piglets (5-day-old), and 2015 had >99.9 % homology in the S gene nucleotide severe diarrhea, vomiting, fecal shedding of virus, and se- sequence, indicating these five viral strains evolved from the vere atrophic enteritis in gnotobiotic pigs (11- to 14-day- same ancestor (Table 2). Further analysis showed that the old) [2, 3]. The data further confirmed that PDCoV were five viruses reported in this study had the highest nucleotide enteropathogenic in pigs. Meanwhile, PEDV or rotavirus identity (98.9 to 99.5 %) with a Jiangxi strain (PDCoV/ showed higher detection rates in PDCoV-positive samples CHJXNI2/2015) isolated from Jiangxi province bordering compared with other TGEV and rotavirus [8, 13–15, 24]. with the northern region of Guangdong province, and As shown above, co-infection of PDCoV and PEDV oc- possessed the lowest nucleotide similarity (95.4 to 95.9 %) curred in nursing piglets (Table 1), indicating that the with PDCoV/Swine/Thailand/S5011/2015 and PDCoV/ diarrhea-related pathogens were quite complex clinically Swine/Thailand/S5015L/2015, isolated from Thailand and not easy to control in the field. Moreover, in the two (Table 2). From the above data, phylogenetic analysis of the recent studies, PDCoV was shown to have higher infectiv- S gene showed that the current PDCoVs circulating in ity in sows with diarrhea (81.0 %, 34/42) than nonclinical southern China were most closely related to other Chinese counterparts (23.5 %, 4/17) [8, 15], which might imply that PDCoV isolates than to those isolated previously from sows often carry PDCoV. And further, it could result in the USA, South Korea and Thailand (Fig. 1). In addition, in the of PDCoV from sows to the foetus and even S gene, any possible recombinant events were not detected newborn piglets, although the pathogenesis mechanism of among those PDCoV strains. PDCoV remains unclear. To further understand the origin of PDCoV, some retro- spective studies were made using PCR and enzyme-linked Sequence comparison and phylogenetic analysis of the N immunosorbent assay (ELISA) [24–26]. In Dong et al. [24] gene of PDCoVs study, 2 of 6 samples collected from Anhui Province of Similarly, N gene sequences of all five PDCoV-positive sam- China in 2004 were positive for PDCoV, up to now, it was ples were identified as 1029 nt in size. Sequence alignment the most ancient time for the detection of PDCoV in results suggested that there was no deletion or insertion in China. Meanwhile, PDCoV could date back to August N gene regions (Additional file 1: Table S1). Consistent with 2013 in United States, where only 3 PDCoV-samples were thedataofSgene,multiplesequencealignmentresultsof detected using PCR in archived samples [25]. As for N gene showed that CH/GD01/2015, CH/GD02/2015, CH/ serology of PDCoV, anti-PDCoV IgG antibodies could GD03/2015, CH/HN01/2015 and CH/GX01/2015 had the date back to 2010 using an indirect anti-PDCoV IgG highest nucleotide homology (99.1 to 99.7 %) with PDCoV/ ELISA based on the putative S1 portion of the spike pro- CHJXNI2/2015, and the lowest nucleotide homology (96.9 tein [26]. The above studies indicated that PDCoV could to 97.1 %) with PDCoV/Swine/Thailand/S5011/2015 have circulated in China at least since 2004 and in United and PDCoV/Swine/Thailand/ S5015L/2015 (Table 2). In States since 2010. Maybe, due to limted samples in the addition, in the phylogenetic tree based on N gene, PDCoVs present study, we did not detect PDCoV in pig samples col- were divided into three main branches (Chinese branch, lected from Guangdong province between 2012 and 2014. American branch and Thai branch). The five viral isolates Although Asian leopard cat coronavirus (GenBank reported from this work were clustered together within the accession no. EF584908) was close to PDCoV in the phylo- Chinese branch (Fig. 2). Moreover, there were no any genetic trees (Figs. 1 and 2), in the future, more Table 2 Nucleotide similarities (%) of S and N genes of our five PDCoV isolates and other reported PDCoVs and coronaviruses Zhai

S gene N gene Journal Virology al. et GenBank No. KU204694 KU204695 KU204696 KU204697 KX534090 KU204698 KU204699 KU204700 KU204701 KX534091 Strain CH/GD01/ CH/GD02/ CH/HN01/ CH/GX01/ CH/GD03/ CH/GD01/ CH/GD02/ CH/HN01/ CH/GX01/ CH/GD03/ 2015 2015 2015 2015 2015 2015 2015 2015 2015 2015 KR131621 PDCoV/CHJXNI2/2015 99.5 98.9 98.9 99.1 99.2 99.7 99.1 99.1 99.3 99.4 KP757892 CHN-JS-2014 99.1 98.5 98.5 98.7 98.7 99.3 99.3 99.3 99.5 99.0 KP757891 CHN-HB-2014 98.9 98.4 98.4 98.7 98.5 98.8 98.6 98.6 98.8 98.5 (2016) 13:136 JQ065042 HKU15-44 98.6 98.2 98.2 98.5 98.3 98.7 98.5 98.5 98.7 98.4 JQ065043 HKU15-155 98.5 98.2 98.2 98.4 98.2 98.7 98.5 98.5 98.7 98.4 KT021234 CH/SXD1/2015 98.4 97.9 97.9 98.1 98.0 99.0 98.8 98.8 99.0 98.7 KT266822 CH/Sichuan/S27/2012 98.4 98.0 98.0 98.2 98.0 99.2 99.0 99.0 99.2 98.9 KM820765 KNU14-04 98.4 97.9 97.9 98.2 98.1 98.8 98.6 98.6 98.8 98.5 KJ620016 MI6148 98.4 97.9 97.9 98.1 98.0 98.8 98.6 98.6 98.8 98.5 KJ584360 MN3092 -a -a -a -a -a 98.8 98.6 98.6 98.8 98.5 KJ584358 PA3148 98.4 98.0 98.0 98.2 98.0 98.8 98.6 98.6 98.8 98.5 KJ584357 KY4813 98.4 97.9 97.9 98.1 98.0 98.8 98.6 98.6 98.8 98.5 KJ584355 IL2768 98.4 97.9 97.9 98.1 98.0 98.8 98.6 98.6 98.8 98.5 KT381613 OH11846 98.4 97.9 97.9 98.1 98.0 98.8 98.6 98.6 98.8 98.5 KJ601779 PDCoV/USA/Illinois 98.4 97.9 97.9 98.1 98.0 98.5 98.3 98.3 98.5 98.3 136/2014 KJ481931 PDCoV/USA/Illinois 98.4 97.9 97.9 98.1 98.0 98.5 98.3 98.3 98.5 98.3 121/2014 KJ769231 OhioCVM1/2014 98.4 97.8 97.8 98.1 98.0 98.3 98.2 98.2 98.3 98.1 KJ601777 PDCoV/USA/Illinois 98.4 97.8 97.8 98.1 98.0 98.7 98.5 98.5 98.7 98.4 133/2014 KJ584359 NE3579 98.4 97.8 97.8 98.1 98.0 98.7 98.5 98.5 98.7 98.4 KJ584356 SD3424 98.4 97.8 97.8 98.1 98.0 98.5 98.3 98.4 98.5 98.3 KJ462462 OH1987 98.4 97.8 97.8 98.1 98.0 98.7 98.5 98.5 98.7 98.4 KJ601778 PDCoV/USA/Illinois 98.3 97.8 97.8 98.0 98.0 98.7 98.5 98.5 98.7 98.4 134/2014 KP995358 OH-FD22 98.3 97.8 97.8 98.0 98.0 -b -b -b -b -b KJ601780 PDCoV/USA/Ohio 98.3 97.8 97.8 98.0 98.0 98.7 98.5 98.5 98.7 98.4 137/2014 ae5o 11 of 5 Page KJ569769 IN2847 98.3 97.8 97.8 98.1 98.0 98.8 98.6 98.6 98.8 98.5 KJ567050 8734/USA-IA/2014 98.3 97.9 97.9 98.1 98.0 98.7 98.5 98.5 98.7 98.4 KP981395 98.3 97.8 97.8 98.0 97.9 98.7 98.5 98.5 98.7 98.4 Table 2 Nucleotide similarities (%) of S and N genes of our five PDCoV isolates and other reported PDCoVs and coronaviruses (Continued) Zhai

USA/IL/2014/026 Journal Virology al. et PDV_P11 KM012168 Michigan/8977/2014 98.3 97.8 97.8 98.0 97.9 98.7 98.5 98.5 98.7 98.4 KP757890 CHN-AH-2004 98.0 97.5 97.5 97.8 97.7 98.1 97.9 97.9 98.1 97.8 KU051641 PDCoV/Swine/Thailand/ 95.8 95.6 95.6 95.9 95.4 97.1 96.9 96.9 97.1 96.8 S5011/2015 (2016) 13:136 KU051649 PDCoV/Swine/Thailand/ 95.8 95.5 95.5 95.9 95.4 97.1 96.9 96.9 97.1 96.8 S5015L/2015 KU984334 P23_15_TT_1115 95.9 95.8 95.6 95.9 95.5 97.8 97.8 97.8 98.0 97.5 EF584908 Guangxi/F230/2006 97.5 97.1 97.1 97.3 97.0 98.0 97.8 97.8 98.0 97.7 JQ065045 HKU17-6124 40.9 40.8 40.8 40.7 40.7 92.9 92.6 92.8 92.9 92.6 FJ376621 HKU12-600 40.7 41.1 41.1 41.0 40.6 76.4 75.7 76.2 76.3 76.3 JQ065044 HKU16-6847 57.0 56.9 56.9 57.0 57.2 75.1 75.3 75.2 75.4 75.1 FJ376619 HKU11-934 65.9 68.9 68.9 66.1 65.8 74.4 74.3 74.5 74.4 74.2 FJ376620 HKU11-796 65.3 65.6 65.7 65.6 65.1 74.4 74.6 74.5 74.4 74.2 KJ408801 OH1414 (PEDV) 38.2 38.3 38.3 38.1 37.8 3.6 3.5 3.5 3.6 3.6 FJ755618 H16(TGEV) 38.1 38.1 38.1 38.0 37.9 2.8 4.9 7.5 5.0 2.8 DQ811787 ISU-1(PRCV) 37.4 25.7 25.7 25.7 37.3 7.3 7.4 7.5 7.4 7.4 BCU00735 Mebus(bovine) 12.4 12.5 12.5 12.4 7.1 2.1 2.1 2.1 2.1 2.1 AY654624 TJF(SARS) 6.8 6.9 6.9 6.8 6.9 8.8 8.8 2.4 8.8 8.1 DQ011855 VW572(PHEV) 21.9 21.9 21.9 21.9 21.8 2.1 2.1 2.1 2.1 2.1 JF893452 YN(CIBV) 22.0 22.1 22.1 22.3 22.0 12.5 12.5 12.5 12.6 12.4 NC_010800 MG10 (Turkey) 24.1 23.5 23.5 23.3 24.0 14.0 14.0 14.0 14.1 14.1 NC_010646 SW1 (whale) 15.0 13.7 13.7 13.8 14.8 7.7 7.7 7.7 7.7 7.7 aS gene of MN3092 was not complete; bS gene of OH-FD22 was not available ae6o 11 of 6 Page Zhai et al. Virology Journal (2016) 13:136 Page 7 of 11

Strains/GenBank No.s Year Genus

Mebus (BCU00735) VW572 (DQ011855) 1972 Betacoronavirus YN (JF893452) 2005 MG10 (NC 010800) TJF (AY654624) 2003 Alphacoronavirus SW1 (NC 010646) Gammacoronavirus OH1414 (KJ408801) 2014 ISU-1 (DQ811787) Alphacoronavirus H16 (FJ755618) 1973 HKU12-600 (FJ376621) 2007 HKU17-6124 (JQ065045) 2007 HKU16-6847 (JQ065044) 2007 HKU11-934 (FJ376619) 2007 HKU11-796 (FJ376620) 2007 Guangxi/F230/2006 (EF584908) 2006 P23 15 TT 1115 (KU984334) 2015 PDCoV/Swine/Thailand/S5011/2015 (KU051641) 2015 PDCoV/Swine/Thailand/S5015L/2015 (KU051649) 2015 CHN-AH-2004 (KP757890) 2004 HKU15-155 (JQ065043) 2010 HKU15-44 (JQ065042) 2009 CHN-HB-2014 (KP757891) 2014 CH/SXD1/2015 (KT021234) 2015 CH/GD01/2015 (KU204694) 2015 CH/GD03/2015 (KX534090) 2015 PDCoV/CHJXNI2/2015 (KR131621) 2015 CH/GX01/2015 (KU204697) 2015 CH/GD02/2015 (KU204695) 2015 CH/HN01/2015 (KU204696) 2015 CHN-JS-2014 (KP757892) 2014 CH/Sichuan/S27/2012 (KT266822) 2012 Deltacoronavirus SD3424 (KJ584356) 2014 OhioCVM1/2014 (KJ769231) 2014 OH1987 (KJ462462) 2014 OH-FD22 (KP995358) 2014 PA3148 (KJ584358) 2014 MI6148 (KJ620016) 2014 8734/USA-IA/2014 (KJ567050) 2014 PDCoV/USA/Ohio137/2014 (KJ601780) 2014 IL2768 (KJ584355) 2014 NE3579 (KJ584359) 2014 PDCoV/USA/Illinois133/2014 (KJ601777) 2014 PDCoV/USA/Illinois134/2014 (KJ601778) 2014 PDCoV/USA/Illinois121/2014 (KJ481931) 2014 PDCoV/USA/Illinois136/2014 (KJ601779) 2014 OH11846 (KT381613) 2014 IN2847 (KJ569769) 2014 KNU14-04 (KM820765) 2014 KY4813 (KJ584357) 2014 Michigan/8977/2014 (KM012168) 2014 0.05 USA/IL/2014/026PDV P11 (KP981395) 2014 Fig. 1 Phylogenetic analysis based on the S gene of PDCoVs and other coronaviruses. Note: Those PDCoV strains from China, America, South Korea and Thiland were labelled using “ ”, “ ”, “ ” and “ ”, respectively. Moreover, PDCoV strains in this study were labelled using left arrows. The collection time was not available for those coronavirus strains labelled using star symbols Zhai et al. Virology Journal (2016) 13:136 Page 8 of 11

Strains/GenBank No.s Year Genus

PDCoV/USA/Illinois133/2014 (KJ601777) 2014 PDCoV/USA/Illinois134/2014 (KJ601778) 2014 KY4813 (KJ584357) 2014 IL2768 (KJ584355) 2014 PDCoV/USA/Ohio137/2014 (KJ601780) 2014 OH-FD22 (KJ584358) 2014 OH1987 (KJ462462) 2014 NE3579 (KJ584359) 2014 8734/USA-IA/2014 (KJ567050) 2014 SD3424 (KJ584356) 2014 Michigan/8977/2014 (KM012168) 2014 USA/IL/2014/026PDV_P11 (KP981395) 2014 OhioCVM1/2014 (KJ769231) 2014 IN2847 (KJ569769) 2014 OH11846 (KT381613) 2014 MN3092 (KJ584360) 2014 MI6148 (KJ620016) 2014 PDCoV/USA/Illinois121/2014 (KJ481931) 2014 PDCoV/USA/Illinois136/2014 (KJ601779) 2014 KNU14-04 (KM820765) 2014 HKU15-44 (JQ065042) 2009 CHN-HB-2014 (KP757891) 2014 Deltacoronavirus CH/SXD1/2015 (KT021234) 2015 CH/Sichuan/S27/2012 (KT266822) 2012 CH/GD01/2015 (KU204694) 2015 CH/GD03/2015 (KX534091) 2015 PDCoV/CHJXNI2/2015 (KR131621) 2015 CHN-JS-2014 (KP757892) 2014 CH/GD02/2015 (KU204699) 2015 CH/GX01/2015 (KU204701) 2015 CH/HN01/2015 (KU204700) 2015 Guangxi/F230/2006 (EF584908) 2006 HKU15-155 (JQ065043) 2010 CHN-AH-2004 (KP757890) 2004 P23_15_TT_1115 (KU984334) 2015 PDCoV/Swine/Thailand/S5011/2015 (KU051641) 2015 PDCoV/Swine/Thailand/S5015L/2015 (KU051649) 2015 HKU17-6124 (JQ065045) 2007 HKU11-934 (FJ376619) 2007 HKU11-796 (FJ376620) 2007 HKU12-600 (FJ376621) 2007 HKU16-6847 (JQ065044) 2007 SW1 (NC_010646) YN (JF893452) 2005 Gammacoronavirus MG10 (NC_010800) OH1414 (KJ408801) 2014 ISU-1 (DQ811787) H16 (FJ755618) 1973 Alphacoronavirus TJF (AY654624) 2003 Mebus (BCU00735) Betacoronavirus 0.5 VW572 (DQ011855) 1972 Fig. 2 Phylogenetic analysis based on the N gene of PDCoVs and other coronaviruses. Note: Those PDCoV strains from China, America, South Korea and Thiland were labelled using “ ”, “ ”, “ ” and “ ”, respectively. Moreover, PDCoV strains in this study were labelled using left arrows. The collection time was not available for those coronavirus strains labelled using star symbols Zhai et al. Virology Journal (2016) 13:136 Page 9 of 11

epidemiological surveys should be warranted to uncover found in CH/Sichuan/S27/2012 [16]. However, for Thai the origin of PDCoV. viral isolates, they owned one additional unique nucleotide At the territory of China, Southern China mainly (C) insertion in the 3’ UTR [17, 18]. The biological signifi- includes Guangdong province, Hainan province and the cance of these naturally occurring deletions or insertions Guangxi autonomous region. Although molecular in PDCoV biology and pathogenesis warrants further detection of PDCoV was performed in these regions investigations. [23, 24], little information was available on PDCoV In this study, five S and five N gene sequences, respect- prevalence. In a study by Chen et al. [23], an overall ively, were obtained to evaluate wherever genetic diversity positive-PDCoV rate of 23.4 % (15/64) was obtained in of PDCoVs existed in southern China. Our results showed all samples collected from Guangdong, Shanxi and that these five S and five N gene sequences were more Hubei provinces. However, more detailed data of closely related to Chinese strains, and all clustered together PDCoV was not available in Guangdong province. inthephylogenetictree(Table2,Figs.1and2).However, Meanwhile, in the study from Dong et al. [24], only CH/GD01/2015 and CH/GD02/2015, reported in this four archived samples from the Guangxi autonomous study, originated from the same pig farm in Guangdong region were examined, but all negative for PDCoV. In province, but had 48 nt and 12 nt differences in the S and this study, we demonstrated that PDCoV circulated and N genes, respectively. The observed 48 nucleotide changes was co-infected by PEDV on those swine farms in in the S gene made these viruses differ by 25 amino acid Guangdong province, Hainan province and the Guangxi residues (Additional file 2: Table S2). For the N gene, the autonomous region, which further contributed to the 12 nucleotide changes among these viruses resulted in 3 epidemiology of PDCoV in these regions despite the amino acid substitutions (Additional file 2: Table S2). relatively low prevalence. Among them, 18 of 25 amino acid differences occurred at the first two-third parts of S gene. Interestingly, in spite of Genetic diversity of PDCoVs amino acid mutation, both S and N protein retained almost The first two reported full-length PDCoV genome se- consistent amino acid properties (especially pH value) quences (HKU15-44 and HKU15-155) were 25, 437 nt (Additional file 2: Table S2). Future study will address im- and 25, 432 nt in length, respectively [1], and they had portant roles of these polymorphisms in viral replication 99.1 % nucleotide similarity with each other. Moreover, and pathogenesis. In addition, they were divided into two further sequence alignment showed a 3-nt (TAA) inser- distinct small branches (Figs. 1 and 2). These findings sug- tion in the S gene and a 3-nt (TTA) insertion in the 3’ gested that PDCoVs in southern China have diverged from untranslated region (UTR) of HKU15-44 [1, 5]. During acommonancestor.Despitetheemerginggeneticdiversity, the past 3 years, PDCoV-associated swine enteric disease overall, PDCoV prevalence is still largely restricted by the was paid great attention in the major pig producing territory as demonstrated in Figs. 1 and 2. countries, especilly United States and China. Up to May For the two enteric coronaviruses (PEDV and TGEV) in 2016, more than 30 complete PDCoV genome sequences pigs, the recombination events were often detected. How- were published in the GenBank database. All were ever, most of them were from intra-recombination [27–30]. generated in China and United States except for one Recently, only one emerging recombinant/chimeric virus sequence from South Korea and three sequences from (named swine enteric coronavirus, SeCoV) was discovered Thailand [8–19]. The Korean strain, KNU14-04, had 25, in swine feces and resulted from inter-recombination of 422 nt in length, with similar genome features (a 3-nt PEDV and TGEV, which had a TGEV backbone and a insertion in the S gene with 3, 483 nt and a 3-nt inser- PEDV spike gene [31, 32]. In this study, there were no any tion in the 3’ UTR, respectively) to all American strains possible recombinant events occurring in PDCoV strains. and the Chinese strain (HKU15-44) [9]. Comparing Maybe, the number and length of our obtained PDCoV se- complete of the remaining Chinese strains to the quences were limited. In the following study, the recombin- American, Thai and Korean counterparts, CHN-HB-2014, ation event of PDCoV warrants further attentions. CHN-JS-2014, PDCoV/CHJXNI2/2015, CH/Sichuan/S27/ 2012, CH/SXD1/2015 and P23_15_TT_1115 only had the 3-nt (AAT) deletion in the S gene (3, 480 nt) [14, 23, 24], Conclusion while CHN-AH-2004 only had the 3-nt (TAA) deletion This study reported the prevalence of PDCoV on swine in the 3’ UTR [5, 24]. In the present study, 3-nt inser- farms in southern China. Phylogenetic analysis of cur- tion was not found in UTR for our five obtained rently circulating PDCoV strains in this region and other PDCoV (data not shown). Moreover, two additional previously reported strains supported the theory of geo- unique features including a 6-nt (TTTGAA) deletion in graphical clustering of PDCoV infection landscape. The the nonstructural protein (nsp) 2 region and a 9-nt origin of various PDCoVs in different countries and re- (GCCGGTTGG) deletion in the nsp 3 region were also gions should be further studied. Zhai et al. Virology Journal (2016) 13:136 Page 10 of 11

Additional files 5. Wang L, Hayes J, Sarver C, Byrum B, Zhang Y. Porcine deltacoronavirus: histological lesions and genetic characterization. Arch Virol. 2016;161:171–5. 6. Zhang Q, Hu R, Tang X, Wu C, He Q, Zhao Z, Chen H, Wu B. Occurrence Additional file 1: Table S1. Sequence information of PDCoVs and and investigation of enteric viral infections in pigs with diarrhea in China. other coronaviruses used in the present study. (DOC 106 kb) Arch Virol. 2013;158:1631–6. Additional file 2: Table S2. AA differences of Spike (S) and Nucleocapsid 7. Homwong N, Jarvis MC, Lam HC, Diaz A, Rovira A, Nelson M, Marthaler D. (N) protein between CH/GD01/2015 and CH/GD02/2015. (DOC 42 kb) Characterization and evolution of porcine deltacoronavirus in the United States. Prev Vet Med. 2016;123:168–74. 8. Hu H, Jung K, Vlasova AN, Chepngeno J, Lu Z, Wang Q, Saif LJ. Isolation and Abbreviations characterization of porcine deltacoronavirus from pigs with diarrhea in the ELISA, enzyme-linked immunosorbent assay; MEGA, molecular evolutionary United States. J Clin Microbiol. 2015;53:1537–48. genetics analysis; N, nucleocapsid; PBS, phosphate buffered saline; PDCoV, 9. Lee S, Lee C. Complete Genome Characterization of Korean Porcine porcine deltacoronavirus; PEDV, porcine epidemic diarrhea virus; PHEV, porcine Deltacoronavirus Strain KOR/KNU14-04/2014. Genome Announc. 2014;2: hemagglutinating encephalomyelitis virus; PRCV, porcine respiratory coronavirus; e01191–14. RDP, recombination detection program; Rota C, porcine rotavirus group C; RT-PCR, 10. Li G, Chen Q, Harmon KM, Yoon KJ, Schwartz KJ, Hoogland MJ, Gauger PC, reverse transcription polymerase chain reaction; S, Spike; SeCoV, swine enteric Main RG, Zhang J. Full-Length Genome Sequence of Porcine coronavirus; TGEV, transmissible gastroenteritis virus; UTR, untranslated region Deltacoronavirus Strain USA/IA/2014/8734. Genome Announc. 2014;2: e00278–14. Funding 11. Ma Y, Zhang Y, Liang X, Lou F, Oglesbee M, Krakowka S, Li J. Origin, evolution, This study was supported by the grants (No. 2013B060500063, No. and virulence of porcine deltacoronaviruses in the United States. MBio. 2014B040404061, No. 2016A040401012 and No. 201508020055) from 2015;6:e00064. Guangdong Provincial Department of Science and Technology and 12. Marthaler D, Jiang Y, Collins J, Rossow K. Complete Genome Sequence of Guangzhou Science Technology and Innovation Commission, respectively. Strain SDCV/USA/Illinois121/2014, a Porcine Deltacoronavirus from the Moreover, this study was also in part supported by USDA/NIFA 2016-67016-24949 United States. Genome Announc. 2014;2:e00218–14. (to D.W.). The funding body was not involved into the design of the study, and 13. Marthaler D, Raymond L, Jiang Y, Collins J, Rossow K, Rovira A. Rapid collection, analysis, and interpretation of data in the manuscript. detection, complete genome sequencing, and phylogenetic analysis of porcine deltacoronavirus. Emerg Infect Dis. 2014;20:1347–50. Availability of data and materials 14. Song D, Zhou X, Peng Q, Chen Y, Zhang F, Huang T, Zhang T, Li A, Huang D, The datasets supporting the conclusions of this article are included within Wu Q, He H, Tang Y. Newly emerged porcine deltacoronavirus associated with the article and two additional files (Additional file 1: Table S1 and Additional diarrhoea in swine in China: identification, prevalence and full-length genome file 2: Table S2). sequence analysis. Transbound Emerg Dis. 2015;62:575–80. 15. Wang L, Byrum B, Zhang Y. Detection and genetic characterization of Authors’ contributions deltacoronavirus in pigs, Ohio, USA, 2014. Emerg Infect Dis. Shao-Lun Zhai and Wen-Kang Wei conceived the project; Shao-Lun Zhai 2014;20:1227–30. designed the experiments; Shao-Lun Zhai and Xiao-Peng Li performed 16. Wang YW, Yue H, Fang W, Huang YW. Complete Genome Sequence of most of the experiments; Xiao-Hui Wen and Dian-Hong Lv contributed Porcine Deltacoronavirus Strain CH/Sichuan/S27/2012 from Mainland China. materials and participated in discussion; Xia Zhou and He Zhang added Genome Announc. 2015;3:e00945–15. the data in the revision version; Shao-Lun Zhai wrote the manuscript; 17. Madapong A, Saeng-Chuto K, Lorsirigool A, Temeeyasen G, Srijangwad A, Feng Li edited the manuscript; Shao-Lun Zhai and Dan Wang supervised Tripipat T, Wegner M, Nilubol D. Complete Genome Sequence of Porcine the work. The final version of the manuscript was approved by all authors. Deltacoronavirus Isolated in Thailand in 2015. Genome Announc. 2016;4: e00408–16. Competing interests 18. 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